The vasculature of the brain comprises specialized endothelial cells that form an intricate network of blood vessels. The formation and healthy functioning of these vessels is supported by other cell types, such as astrocytes and pericytes, that interact with the endothelium through direct contact and secreted factors. Together, these cell types form the blood-brain barrier (BBB). This barrier maintains a homeostatic environment for the brain and is often disturbed in brain disorders, such as neurodegenerative- and neuroinflammatory disorders.
A physiologically relevant BBB model (1) comprises the different BBB cell types, (2) shows the formation of adherens and tight junctions, (3) forms a tight barrier, (4) and presents with functional BBB transporters. To add complexity, neurons can be added to model the full neurovascular unit and study not only how drugs affect the BBB and if they cross the BBB, but also what their effect is once they’ve crossed the barrier and reach the brain.
Figure: Confocal image animation of a co-culture with primary astrocytes and HUVECs.
We describe a primary human model of the blood-brain barrier (BBB) comprising a microvessel of brain endothelial cells, supported by astrocytes and pericytes. The model shows expression of adherens- and tight junction proteins, barrier formation, and functional P-gp transport.
The OrganoPlate® 3-lane comprises 40 microfluidic chips that can be used to culture miniaturized tissues and organs. (b) Phase contrast image of BBB on-a-chip in the OrganoPlate® 3-lane. The endothelial microvessel (top channel) is grown against an extracellular matrix gel (middle channel). Astrocytes and pericytes are added to the bottom channel and migrate through the gel to the endothelial vessel. Scale bar is 100 μm. (c) Fluorescent image showing a BBB culture of endothelial cells (red) and astrocytes and pericytes (green). Scale bar is 100 μm. (d-e) Staining of the endothelial microvessel for adherens junction markers (d) PECAM-1 and (e) VE-cadherin and tight junction markers (f) Claudin-5 and (g) ZO-1. Scale bars are 50 μm.
Barrier integrity assay
Barrier function of the BBB model can be assessed using a real-time barrier integrity assay. In this assay, a fluorescent dye is perfused through the lumen of the model’s endothelial vessel. In case of a leak-tight barrier, all dye is retained in the microvessel (figure 2a), while in case of a leaky barrier or a cell-free control, the fluorescent dye leaks into the adjacent ECM gel channel (figure 2b-c). The fluorescent intensity in both channels is monitored over time and is used to quantify barrier function in each condition (figure 2d). Figure 2e shows that when the BBB model is treated to mimic ischemic stroke, its barrier function strongly decreases compared to control.
A different assay was employed to assess the function of one of the BBB’s main efflux transporters, the P-glycoprotein (P-gp) transporter. Calcein-AM was perfused through the lumen of the BBB’s endothelial vessel and taken up by the cells, making the cells green-fluorescent. In presence of a P-gp inhibitor, P-gp efflux of calcein is reduced, resulting in accumulation of green-fluorescent signal inside the cells (figure 3a). The endothelial cells forming the model’s barrier were imaged using a FITC filter and the green-fluorescent signal per cell was calculated using an additional Hoechst staining to visualize the nuclei. Figure 3b shows that in presence of a P-gp inhibitor, the endothelial cells of the BBB model show increased green- fluorescence, indicative of functional P-gp transport.
This work was partly supported by the European Union under grant agreement No 667375 (CoStream) and No 115975 (Adapted).